Maddie Ceminsky
Ceminsky, Maddie
Year: 2022
Faculty Advisor: David Stern
Mentor: Kevin Baudry

“Toward a Fine Characterization of Chloroplast Ribonuclease J in Arabidopsis

Project Summary:

The chloroplast contains around 100 genes and has elements of both prokaryotic and eukaryotic gene expression. Transcription in chloroplasts has inefficient termination, resulting in polycistronic and some monocistronic transcripts, necessitating large-scale maturation such as RNA processing. Ribonucleases play key roles in mediating these steps; endonucleases cleave RNA at internal sites and exonucleases process RNA extremities.

In vitro, RNase J (RNJ) is the only plastidial ribonuclease with 5’→3’ exonuclease activity and it also carries endonuclease activity. Land plant RNJs contain a GT-1 domain at the C-terminus, a domain generally found in transcription factors; its function in RNJ remains unknown. RNJ knockout is embryo lethal in land plants and virus-induced gene silencing (VIGS) of RNJ results in an accumulation of antisense RNA (asRNA), which prevents translation. This suggests a role for RNJ in RNA quality control by degrading asRNA. The relative in vitro exonuclease and endonuclease activity of RNJ differs by species: Arabidopsis thaliana RNJ (AtRNJ) has robust endonuclease and minor exonuclease activity, bacterial Bacillus subtilis RNJ1 (BsRNJ1) mainly exhibits exonucleolytic activity, and the green algae Chlamydomonas reinhardtii RNJ (CrRNJ) exhibits exclusively endonucleolytic activity. The structural basis underlying this differential activity is unknown.

Here, we attempted to decipher the functions of RNJ in Arabidopsis plastid. We created lines expressing AtRNJ and CrRNJ under the Arabidopsis native promoter in an rnj mutant background. In addition, we started work to create lines expressing BsRNJ1 under the Arabidopsis native promoter and AtRNJ under an embryo specific promoter to bypass mutant embryo lethality in the same background. In parallel, we expressed 35S::YFP:GT-1 in wildtype plants. Preliminary results show that T1 plants of this line develop albino leaves on soil.

My Experience:

Over the past 10 weeks, my fantastic mentor Kevin Baudry has helped me to grow as a scientist in a variety of ways. I have developed my abilities to plan and manage experiments, think like a scientist, and troubleshoot difficulties. Additionally, I was exposed to plant biology on all scales, from molecular level to plant phenotype, giving me a greater appreciation for the dramatic impact of small changes to the genome. Through this experience, I have gained confidence in myself as a scientist and in my ability to further my scientific career. This fall, I will take the exciting step of applying to graduate school in biochemistry or molecular biology.